Calcium plays an indispensable role in cell permeability, the formation of bones and teeth, blood coagulation, transmission of nerve impulse, and normal muscle contraction. The concentration of calcium ions in the blood is, along with calcitrol and calcitonin, regulated mainly by parathyroid hormone (PTH). Although calcium intake and excretion may vary, PTH serves through a feedback mechanism to maintain a steady concentration of calcium in cells and surrounding fluids. When serum calcium lowers, the parathyroid glands secrete PTH, affecting the release of stored calcium. When serum calcium increases, stored calcium release is retarded through lowered secretions of PTH.
The complete form of human PTH, sometimes referred to in the art as hPTH but referred to in the present invention as an example of PTH agonist, is a unique 84 amino acid peptide (SEQ ID NO:1), as is shown in FIG. 1. Researchers have found that this peptide has an anabolic effect on bone that involves a domain for protein kinase C activation (amino acid residues 28 to 34) as well as a domain for adenylate cyclase activation (amino acid residues 1 to 7). However, various catabolic forms of clipped or fragmented PTH peptides also are found in circulation, most likely formed by intraglandular or peripheral metabolism. For example, whole PTH can be cleaved between amino acids 34 and 35 to produce a (1–34) PTH N-terminal fragment and a (35–84) PTH C-terminal fragment. Likewise, clipping can occur between either amino acids 36 and 37 or 37 and 38. Recently, a large PTH fragment referred to as “non-(1–84) PTH” has been disclosed which is clipped closer to the N-terminal end of PTH. (See R. LePage et al., “A non-(1–84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples,” Clin. Chem. (1998); 44: 805–810.)
The clinical need for accurate measurement of PTH is well demonstrated. Serum PTH level is one of the most important indices for patients with the following diseases: familial hypocalciuria; hypercalcemia; multiple endocrine neoplasia types I and II; osteoporosis; Paget's bone disease; primary hyperparathyroidism—caused by primary hyperplasia or adenoma of the parathyroid glands; pseudohypoparathyroidism; and renal failure, which can cause secondary hyperparathyroidism.
PTH plays a role in the course of disease in a patient with chronic renal failure. Renal osteodystrophy (RO) is a complex skeletal disease comprising osteitis fibrosa cystica (caused by PTH excess), osteomalacia—unmineralized bone matrix (caused by vitamin D deficiency), extraskeletal calcification/ossification (caused by abnormal calcium and phosphorus metabolism), and adynamic low bone turnover disease (contributed to by PTH suppression). Chronic renal failure patients can develop RO. Failing kidneys increase serum phosphorus hyperphosphoremia) and decrease 1,25-dihydroxyvitamin D (1,25-D) production by the kidney. The former results in secondary hyperparathyroidism from decreased gastrointestinal calcium absorption and osteitis fibrosa cystica from increased PTH in response to an increase in serum phosphorus. The later causes hypocalcemia and osteomalacia. With the onset of secondary hyperparathyroidism, the parathyroid gland becomes less responsive to its hormonal regulators because of decreased expression of its calcium and vitamin D receptors. Serum calcium drops. RO can lead to digital gangrene, bone pain, bone fractures, and muscle weakness.
For chronic renal failure patients with secondary hyperparathyroidism, a number of different therapeutic treatments are available. One can administer calcium carbonate so as to directly adjust the available calcium ion level. However, with the increasing incidence of ectopic calcification, increasing calcium intake is often not desirable. One can administer calcimimetics, such as AMG073 made by Amgen, Inc. of Thousand Oaks, Calif. However, AMG073 has not been approved for use in the USA. One can administer vitamin D analogues, (such as the Calcijex® or Zemplar® brands made by Abbott Labs of Abbott Park, Ill.; Rocaltrol brand made by Roche Laboratories of Basle, Switzerland; Oxarol® brand made by Chugai Pharmaceutical), so as to lower PTH. However, researchers have found that vitamin D analogues can oversuppress PTH, thereby leading to adynamic low bone turnover disease setting the patient at risk of ectopic and vascular calcification. (See the package insert for Zemplar, Abbott Reference 06-9998-R1-Rev, April 1998. See the package insert for Rocaltrol®, Roche Laboratories, inc. November 1998 Product identification Guide, page 334.)
Researchers have also found that a large circulating PTH fragment (e.g., cyclase inactive parathyroid hormone) functions as a naturally occurring PTH antagonist. Cyclase inactive PTH has been found to be useful, alongside whole PTH, as an indicator in separating untreated end stage renal disease (ESRD) patients with high bone turnover from those with adynamic low bone turnover. (See Faugere, M. C. et alia. “Improved Assessment of Bone Turnover by the PTH 1–84/largeC-PTH fragments ratio in ESRD patients”, Kidney International 2001; 60: 1460–1468.) Moreover, researchers have found that cyclase inactive PTH can cause adynamic low bone turnover by inhibiting the formation of osteoclasts, bone resorption, and bone turnover. (See Divieti, P. et alia, “In vitro Inhibition of Bone Resorption by Human PTH (7–84)” J. Bone Miner Res 2001:Suppl 1, S307. See also Faugere, M. C. et alia, “The Effects of PTH (1–84) on bone turnover are Antagonized by PTH (7–84) in Thyroparathyroidectomized and Nephrectomized Rats”; J Am Soc Nephrol 12:2001, 764A.)
Determining circulating biologically active PTH levels in humans has been challenging. One major problem is that PTH is found at low levels, normally 10 pg/mL to 65 pg/mL. Coupled with extremely low circulating levels is the problem of the heterogeneity of PTH and its many circulating fragments. In many cases, immunoassays have faced substantial and significant interference from circulating PTH fragments. For example, some commercially available PTH kits have almost 100% cross-reactivity with the non-(1–84) PTH fragment, (see the LePage article).
PTH immunoassays have varied over the years. One early approach is a double antibody precipitation immunoassay found in U.S. Pat. No. 4,369,138 to Arnold W. Lindall et alia. A first antibody has a high affinity for a (65–84) PTH fragment. A radioactive labeled (65–84) PTH peptide is added to the sample with the first antibody to compete for the endogenous unlabeled peptide. A second antibody is added which binds to any first antibody and radioactive labeled PTH fragment complex, thereby forming a precipitate. Both precipitate and supernatant can be measured for radioactive activity, and endogenous PTH levels can be calculated therefrom.
In an effort to overcome PTH fragment interference, immunoradiometric two-site assays for intact PTH (I-PTH) have been introduced, such as Allegro® Intact PTH assay by the Nichol's Institute of San Juan Capistrano, Calif. In one version, a capture antibody specifically binds to the C-terminal portion of hPTH while a labeled antibody specifically binds to the N-terminal portion of the captured hPTH. In another, two monoclonal antibodies were used, both of which attached to the N-terminal portion of hPTH. Unfortunately, these assays have problems in that they measure but do not discriminate between whole PTH and non-whole PTH peptide fragments. This inability comes to the fore in hyperparathyroid patients and renal failure patients who have significant endogenous concentrations of large, non-whole PTH fragments.
Recently, Scantibodies Laboratory, Inc. of Santee, Calif. USA introduced a series of novel kits that allow for the accurate measurement of both PTH agonist and PTH antagonist. The PTH agonist assay is a direct measurement, while the PTH antagonist assay is a calculated value from the difference of the PTH agonist assay value and a total PTH (including both PTH agonist and PTH antagonist) assay value. A number of unexpected advantages have become available to the physician, including the first non-invasive method for assisting in the differentiation of secondary hyperparathyroid patients with HBT and ALBT.